BIOC0003

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Ratio of fluorescence with A/without A

1/1+(R₀/R)⁶, where R₀ is distance at which half F lost

Kw at 25 degrees

10^-14

[H+] from pH

10^-pH

A260 and OD

1A260 unit is amount of nucleic acid in 1ml that gives 1OD

Pure DNA

260/280 of 1.8

Pure RNA

260/280 of 2.0

Absorbance value of Nucleic Acids

260nm

ssDNA A260

33microg

Oligonucleotides A260

37microg

RNA A260

40microg

dsDNA A260

50microg

To find conc of two components in a mixture from absorbance

A = ([A]xEa x l) + ([B] x Eb x l) for each wavelength, do simultaneous equations

Beer Lambert Law

A = Ecl

Photons in UV/vis/near IR

Cause electronic transitions if enough energy

Chemiluminescence

Chemical reaction excites electrons

Bioluminescence

Chemiluminescence in biological system

GFP structure

Chromophore surrounded by antiparallel beta can which excludes solvent molecules to avoid quenching

Circular dichroism

Difference between absorption of L-CPL and R-CLP

Stokes shift

Difference in intensity of absorbed and emitted light

Prism

Different wavelengths travel at different speeds, longer wavelength (red) refracted less, high efficiency, ~100% transmittance

FRET

Donor absorbs photon, and energy of excited state transferred to Acceptor which then releases a proton, resulting in an increase in A and decrease in D fluorescence

Cons of GFP

Large domain may disturb protein structure

States and fluorescence

Light emitted when S1 state returns to S0 state, light stops being emitted 10⁻⁸s after absorbed

Optical Dichroism

Log Incident/Transmitted

Effect of stray light on absorbance

Log[(I₀+s)/(I+s)]

Emission spectra vs absorption

Longer wavelength, lower energy

Proteins affects on 260/280

Lowers

Units of E

M-1cm-1

Diphenylhexatreiene

Measures physical states of lipid bilayers via anisotropy, detects detergent micelle formation; very hydrophobic, partitions into lipid bilayer with fluorescence indicating bilayer fluidity; lots of conjugated bonds, planar as has resonance structures

Energy of wave

hv = hc/λ

Henderson-Hasselbalch equation

pH = pKa + log [A-]/[HA]

Speed of light

λv, 3e8 m/s

Gibbs, enthalpy and entropy

∆G = ∆H- T∆S

∆G when protons involved

∆G°', remove [H⁺] from Ke, quote pH

∆G from ∆G⁰

∆G⁰ -RTlnQ

Gibbs and equilibrium

∆G⁰=-RTln(Ke)

∆G of cell

−nFE

Countering effect of equipment on emission

Generate correction factor from known standard

Fluourescence

Absorbance of a photon excites an electron, and some of the energy is remitted as light with a longer wavelength

Follow rate of reaction using absorbance spectrometry

Absorbance vs time

Absorbance of multiple species

Additive

Joule

Amount of energy expended when 1 N applied through 1m

∆G⁰ definition

Amount of energy harnessed to do useful work by a reaction starting under standard conditions

pH from [H+]

-log[H+]

Relationship between intensities and absorbance

A= log[I₀/I]

Plot of absorbance vs conc

At high conc, greater difference between ideal and actual behaviour so line stops being straight

Why is the signal measured at 90° to excitation

Avoid detection of unemitted light, possible as fluorescence emitted in all directions

Using an electrode to measure concentration

Barrier between two electrodes only permeable to one ion type, ion causes a pd across barrier related to ion gradient, potential measured between two electrodes changes, calibrate to provide conc readout

F group isolated from/exposed to water

Blue/red shift

Calibration curve of

Conc vs absorbance

If two substances can be interconverted, absorbance at isosbestic point

Constant regardless of conc

Spectrophotometer detector

Converts photon to electron and measures current, can be photomultiplier or photodiode

Effect of equipment on excitation

Correct effect of differential lamp strength vs wavelength by splitting small amount of excitation light and sending it through a secondary standard solution and detector with wavelength independent response

Covalent fluorescence reactive group X

Cysteine reagents, Lyseine reagents

Nernst equation

Eh = Em + R.T./nF x ln[Ox]/[Red] (n is moles of electrons)

Gram/small calorie

Energy needed to increase 1g of water by 1°C

Non covalent fluorescent probes

Ethidium bromide,diphenylhexatriene

Beers Law Fluorescence

F ~ E l c Qy

Covalent fluorescent probes

Fl group (F) attached to chemically reactive group (X) which reacts with protein or is directly attached to biomolecule

Buffer in blood

H2CO3/HCO3-

pKa from titration

Half of end point, pH when [A-] = [HA]

Inner filter effect

Higher conc of fluorescent molecules decreases proportion of light interacting with molecule of interest; linear relationship between F and C tails off at high c

Properties of fluorescent compounds

Highly conjugated, condensed system of fused rings, electron donating groups, rigid, planar

Standard hydrogen electrode

H⁺ + e⁻ ⇌ 1/2H₂ (g)

Effect of stray light at high absorbency

Increases as less light transmitted

Action spectra

Indirectly monitor a process, eg photosynthesis depends on light absorption

Emission spectra

Intensity of em vs wavelength of em; gives colours of light, measure using emission monochromator

Excitation spectra

Intensity of em vs wavelength of ex, vary excitation monochromator, gives coloursof light able to cause excitation

Scintillation

Interaction with radioactive particle excites electron

Ethidium bromide

Intercalates between DNA bases; large, largely planar, many conj bonds

Pros of GFP

Non toxic, can be used on whole organism

Eh

Overall cell potential

Colours of haemoglobin

Ox- brown, red- purple, O2 bound red- red

Lower E

Oxidised

Vibrational splitting

Photons absorbed excite electrons to higher states, which then trickle down to emit photon at lower energy and longer wavelength; all excited vibrational levels return to ground state prior to emission

Solvent effects

Polar solvent rearranges self to stabilise and lower energy of system, electron transitions occur too quickly for this to occur immediately but overall solvent interactions lower the energy of the states

Fluorescence Activated Cell Sorting

Primary antibody binds to cell epitope,second fluorescent antibody binds to constant region of first, flow cytometer sorts via electrostatic deflection

Dispersion element types

Prism or diffraction grating

Uses of covalent fluorescent probes

Probe local environment of individual amino acid residue, monitor conformational changes, determine membrane protein topography, detect molecule location in cell, determine abundance of CSM, FACS

Fluorescence and conc of fluorophore

Proportional with exception of stray light

Collisional quenching

Random non interactive collisions deactivate excited state, this type requires near contact

Quantum yield Qy

Ratio of emitted to absorbed photons, range from 0-1, number of events triggered per photon absorbed

Electrode potential of cell

Red-ox

Cathode

Reduction

Static quenching

Reporter and quencher form intramolecular dimer, non flu ground state complex forms

Monochromator

Selects wavelength of light

Source of fluorescence in GFP

Ser 65- Tyr 66- Gly 67 specially bonded on alpha helix

Isosbestic point

Wavelength at which two species have the same E

Spectrophotometers

Source, entrance slit, dispersion device, monochromator, sample, detector

Circular Dichroism Set up

Source, monochromator, linear polariser, modulator, sample, detector

Em

Standard cell potential

Quenching types

Static and Dynamic/collisional

Diffraction grating

Surface with closely spread grooves, splits and diffracts light, groove period must be on order of desired wavelength, constant dispersion

Phosphorescence

T1 returns to S0 state, as T1 is long lived, emission can occur after source turned off

Intrinsic fluorescence

Trp, Tyr, somewhat Phe

Features of a good quencher

Unpaired electrons, heavy atoms (affect electron spin spin state, increase S1 to T1 transition), electron transfer event

Reasons for differences in emission vs absorption

Vibrational splitting, solvent effects

Spectrofluorometer

Xe lam, excitation monochromator, sample, 90° to emission monochromator, detector

Kw equation

[H+][OH-]


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